A Quantitative Method for the Differential Determination of Hydroxylamine and BetaAspartyl Hydroxamate in Mixtures JACOB YASHPHE, YEHESKEL S. HALPERN, and NATHAN GROSSOWICZ Department of Bacteriology, The Hebrew University, Hadassah Medical School, Jerusalem, Israel
b A method for the differential estimation of hydroxylamine and @-aspartyl hydroxamate in mixtures i s based on the known color reaction between nitrite, sulfanilic acid, and a-naphthylamine. The substances tested are oxidized by iodine to nitrite. Whereas hydroxylamine i s oxidized at pH values of 2.3 and 3.7, the hydroxamate reacts only at pH 3.7. Thus, a differential determination of the two substances can be made by performing the test at both pH levels. For the quantitative recovery of hydroxamates it i s essential to perform the oxidation at pH 3.7. At this pH, however, the color reaction i s slower; to speed up color development the pH i s lowered to 2.8 after oxidation of the hydroxamate i s completed. Similar results are obtained with a variety of hydroxamates other than @-aspartyl hydroxamic acid. The method should aid in the detection of hydroxamates in biological systems where hydroxylamine i s an intermediate.
A
METHOD for the determination of hydroxylamine is based on the observation of Blom ( 2 ) that hydroxylamine is oxidized quantitatively to nitrite by iodine in a n acetic acid medium and the nitrite thus formed is estimated by the color reaction with sulfanilic acid and a-naphthylamine. Hydroxamic acids are generally determined by their color complexes 1%-ithferric chloride (6). This method, however, is not very sensitive. When quantities are very small the hydroxamate must be hydrolyzed and the reCOMJlON
Table I.
METHODS
Required Solutions. A, ,570 sodium acetate; B, sulfanilic acid reagent (10 grams of sulfanilic acid per 1 liter of 30% acetic acid) ; C, iodine reagent (1.3 grams of iodine per 100 ml. of glacial acetic acid); D, 2.5% sodium thiosulfate; E, a-naphthylamine reagent (3 grams of l-naphthylamine per 1 liter of 30y0 acetic acid); F, 0.1M acetate buffer, pH 3.6; G, concentrated hydrochloric acid (specific gravity 1.165 grams) diluted 1 t o 4 with distilled water. Estimation of ,%Aspartyl Hydroxamic Acid. To 1 ml. of sample add 5 nil. of A, 1 ml. of B, and 0.5 ml. of C. Shake t h e mixture and allow i t t o stand for 3 to 5 minutes. Remove t h e excess iodine by adding 0.5 ml. of D ; add 0.5 ml. of G, a n d stir t h e contents. T h e color is brought about by t h e addition of 1 ml. of a-naphthylamine ( E ) ; make the volume 10 ml., and read after 20 minutes in a Coleman Junior spectrophotometer at 530 mp. Carry out the ivhole pro-
Oxidation Ratesof Hydroxylamine and p-Aspartyl Hydroxamate by Iodine (Reaction carried out at 15' C . )
blin.
pH 2.3
Absorbance &Aspartyl Hydroxamate, 0.1 pJI pH 3.7 pH 2.3 pH 3.7
2
0.400 0.400 0.400 0.385 0,350 0.260
0.400 0.410 0.400 0.385 0.360 0.290
Time of Reaction, 5 10 20 30 60
518
leased hydroxylamine estimated by Blom's method ( 3 ) . Bergmann and Segal ( 1 ) found that hydroxamates could be oxidized by iodine without prior hydrolysis. However, in a previous work ( 4 ) ,while using the method of Bloin (3) for the estimation of hydroxylamine in the presence of p-aspartyl hydroxamic acid, the latter was found not to be oxidized. This discrepancy led to a more thorough investigation of this method. I n the course of these studies it was found possible t o determine hydroxylamine and hydroxamates in mixtures simultaneously.
NHZOH, 0.1 p
ANALYTICAL CHEMISTRY
M
0 0 0
0.400 0.400 0.390 0.370 0 330 0,275
cedure a t a temperature of 10' to 15' C. Estimation of Hydroxylamine. Use the same procedure except replace solution A by 5 ml. of F. The concentrations of hydroxylamine and hydroxymates suitable for colorimetric determination lie betn-een 0.02 and 0.1 pilf per sample. EXPERIMENTAL
I n f l u e n c e of pH. K h e n the methods of Blom ( 2 ) and Bergmann and Segal ( 1 ) were compared, the only significant difference between the two mas the p H of the reaction mixture. After addition of the iodine solution, the p H was 2.1 by the method of Blom and 3.7 by that of Bergmann and Segal. Therefore, the effect of p H on the conversion of p-aspartyl hydro\;amate and hydroxylamine to nitrite by oxidation with iodine IT as studied. The readings nere made 20 minutes after the addition of a-naphthylamine, according to Blom's method (a). As shown in Figure 1, the color reaction of p-aspartyl hydroxamate is strongly dependent upon the hydrogen ion concentration. The maximal color intensity is obtained a t p H 3.7. The reaction rate declines sharply n i t h a slight change in p H on either side. At p H 2.0 to 2.3, practically no color develops. Similar results are obtained when succinyl hydroxamic, y-glutamyl hydroxamic, lacthydro\amic, acetohydroxamic, cinnamohydroxamic. salicyl hydrosamic, and benzohydroxamic acids are used instead. Oxidation Rates of Hydroxylamine
and p-Aspartyl Hydroxamate by Iodine. The oxidation reaction of
both substrates proceeds a t a very fast rate and is completed after 2 minutes, t h e shortest time interval used. On t h e other hand, exposure t o iodine for 10 or more minutes results in lower color values. Therefore, a n oxidation time of 3 t o 5 minutes was chosen (Table I). Changes in Color Intensity with Time. Figure 1 s h o w t h a t different
p H conditions affect t h e rate of color formation. At p H 2.3 t o 3.0, t h e maximal color intensity with hydroxylamine is obtained within 20
0.400.350.30 W
2 a
0.25-
m
8 v,
m
0.20-
a
0
0l I/, I
L4
1.8
2.2
2.6
3
0.04
0.06
0.I
0.08
@M Figure 2. Calibration curves of nitrite, hydroxylamine, and @-aspartylhydroxamic acid (each 0.1 p M )
f
0.10
0.02
0
Nitrite Hydroxylamine 0 &Aspartyl hydroxamic acid Tests performed at p H 3.7 and temperature of 15' C.; calibration curves of nitrite and hydroxylamine at p H 2.3 identical to those at p H 3.7; p-aspartyl hydroxamic acid gave no color reaction at pH 2.3
0
3.4
34
4.2
4.6
5
PH Figure 1. Influence of pH on color formation from nitrite, hydroxylamine, and &aspartyl hydroxamic acid [each
0.1 p M ) 0 Nitrite Hydroxylamine 0 /3-Arportyl hydroxamic acid Readings obtained 2 0 minutes after addition of a-naphthylamine --- Readings obtained 90 minutes after addition of reagent Reaction carried out at 15' C.
_--_
minutes, while a much longer time is required for the complete color development at p H 3.7. There is no color development with p-aspartyl hydroxamic acid even after 4 hours, a t p H values below 2.4. On the other hand, a t p H 3.7 maximal values are obtained after approximately 90 minutes. The reaction described is a two-step process consisting of oxidation of the hydroxylamine or hydroxamate to nitrite, and conversion of the latter to a colored substance. 130th oxidation and color formation with hydroxylamine proceed rapidly to completion a t the lower p H (2.3) (Figure 1). Because the second step is the same regardless of the nature of the substrate, it was felt that acidification of the reaction mixture to p H 2.8 after oxidation was completed would lead to a rapid color development. This was proved to be true. When nitrite was used as a substrate the color reaction slowed down with increase of p H (Figure 1). Thus, the second step controls the rate of the color formation. Identical values were obtained whether the reaction mixture was left for 90 minutes a t p H 3.7 or for only 20 minutes after acidification to p H 2.8. Effect of Temperature. Results are best Lvhen the reaction is carried out a t 10' to 15' C. At higher tem-
peratures p-aspartyl hydroxamate reacts t' Some extent even at pH 2*3* Temperatures below loo c* reduce the rate of the reaction. Of
Calibration Curves for Estimation and pHydroxamic Acid. Figure
shoWs the 'lopes Of the "libration curves of nitrite, hydroxylamine, and hydroxamic acid at pH 3.7. Eauimolar quantities of each of the three substances give identical color intensities. These results show t h a t both hydroxylamine and p-
aspartyl hydroxamic acid are converted quantitatively to nitrite, the substrate responsible for the color reaction. Thus, the same calibration curve for the estimation of each of the three compounds can be used. Estimation of Mixtures of H y droxylamine and B-Aspartyl Hydroxamic Acid. From the above data
i t was inferred t h a t the color intensity obtained with a mixture of the two comuounds a t D H 3.7 should be equal t o the sum of the color intensities given by the same amounts of each of the tivo substances \vhen determined separately. On the hand, a t p H 2.3 it would represent only the amount of in the mixture. The difference between these two values T,.ould gire the amount of the hSdroxamate present, The results of the experiment described in Table I1 confirm the validity of this assumption. DISCUSSION
The
differential
estimation
of
Table II.
Differential Determination of Mixtures of Hydroxylamine and @-Aspartyl Hydroxamic Acid (Reaction carried out a t 1.5' C.) KH,OH. @-Aspartyl Molar HCl Hydroxamic Percentage Total Recovery, 7 0 Absorbance0 Added, Acid Added, of T\",OH ,UM pM in Mixture p H 2.3 p H 3.7 pH 2.3 p H 3.7 0.02 0.02 50.0 50.0 0.080 0.150 93.7 34.5 0.02 0.04 33.3 0.085 0.240 100.0 25.0 0.02 0.06 25.0 0.080 0.320 100.0 22.5 0.02 0.08 20.0 0.090 0.390 97.5 0.04 0.02 66.7 70.8 108.3 0.170 0.260 0.04 0.04 50.0 50.0 103.1 0.160 0.330 42.5 0.04 0.06 40.0 0.170 0.400 100.0 0.06 0.02 106.2 81.2 75.0 0.260 0.340 0.06 0.04 60.0 100.0 65.0 0.260 0.400 0.08 0.02 80.0 102.5 85.0 0.340 0.410 a Measured 90 minutes after the addition of or-naphthylamine.
VOL. 32,
NO. 4, APRIL 1960
519
hydroxylamine and hydroxamates in mixtures, as outlined in this paper, is based on the fact that hydroxylamine gives a color reaction by Blom’s method, both a t p H 2.3 and 3.7, and the hydroxamates react only a t p H 3.7. Because nitrite, the oxidation product of the two substances, gives full color a t both pH values, it follows that hydroxamate can only be ovidized by iodine to nitrite under less acid conditions. Therefore, to obtain a quantitative oxidation of the hydroxamate, it is essential to secure and maintain a p H of 3.7 for the duration of the oxidation reaction. K i t h hydroxylamine, the oxidation reaction is not impaired by the high hydrogen ion concentration (pH 2.3). The differences in the electrolytic dissociation of the two substances perhaps explain this phenomenon. Thus, a t p H 3.7 we get the total amount of hydroxylamine and hydroxamate present in the mixture, while by performing the same
procedure at p H 2.3, only hydrosylamine is being determined. However, while the color development a t p H 2.3 is rapid, the reaction rate is considerably slower a t pH 3.7. Kevertheless, the reaction with hydroxamate can proceed as rapidly as that of hydroxylamine if the pH of the reaction mixture is lowered to 2.8 after osidation. Calibration curves for nitrite, hydroxylamine, and 0-aspartyl hydroxamic acid (Figure 2) show that equivalent amounts of the three substances give practically the same color intensities, indicating that the oxidation of hydroxylamine and hydroxamate to nitrite is quantitative. This justifies the use of the method suggested here. The amount of hydroxamate present in a mixture is obtained by subtracting the amount of hydroxylamine found a t p H 2.3 from the total amount of nitrite determined a t p H 3.7. The simultaneous determination of
hydroxylamine and hydroxamate should be valuable in studies of biological oxidation of ammonia ( 5 ) , nitrite reduction (Y),and other reactions where, in addition to hydroxylamine, hydroxamates may be formed as intermediary metabolites. LITERATURE CITED ( 1 ) Berginnnn, F., Segal, R., Biochem. J . 6 2 , 5 4 2 (1956). ( 2 ) Blom, J., Ber. deut. chem. Ges. 50, 121 (1926). ( 3 ) ,Feigl, F., “Spot Tests,” Vol. 2, Elsevier, London, 1954. (4) Grossowicx, N., Halpern, Y. S., J . B i d . Chem. 228, 643 (1957).
( 5 ) Hofman, T., Lees, H., Biochem. J .
54,579 (1953). ( 6 ) Lipmann, F., Tuttle, L. C., J . Rid. Chem. 159, 21 (1945). ( 7 ) Zucker, If.,Yason, A , , Ihid., 213, 463 (1R55).
RECEIVEDfor review J ~ l y 14, 1958. Accepted Kovember 23, 1959.
Adsorption of the Elements from Hydrofluoric Acid by Anion Exchange J. P. FARIS Argonne National laborafory, lernonf, 111.
b Elution characteristics for some 50 elements in a hydrofluoric acid medium were studied with a strongly basic anion exchange resin. Estimates of distribution coefficients in 1M to 24M acid were obtained by spectrographic analysis of column effluent solutions. The adsorption data observed for elements in this medium suggest many possible analytical separations.
W
ITHIX recent years the availability of stable high capacity ion exchange resins has led t o the rapidly increasing use of these materials in the analytical field. The added emphasis on ion eschang? methods of separation is evidenced by the number of detailed quantitative procedures and variety of applications being presented in the literature. The various materials available, the techniques employed, and development of the theory are described in the manufacturers’ literature, periodical review articles, and several recent books ( 7 , 9 ) . Anion exchange resins have proyed to be extremely useful for performing rapid quantitative separations. The comprehensive survey of adsorption characteristics in a hydrochloric acid
520
ANALYTICAL CHEMISTRY
medium presented by Kraus and Kelson (6) has resulted in a variety of analytical applications. Anion exchange data have also been presented for a number of the metals in nitric acid ( 1 , 2, 6, IS) and hydrochloric-hydrofluoric acid mixtures (5, IS). Hydrofluoric acid (IO), sulfuric acid (2, 5 ) , and phosphoric acid ( 4 ) systems have been reported less extensively. To extend the scope of anion euchange-analytical separation methods and also to provide a basis for planning future experiments in acid mixtures, a general survey of the metals in a hydrofluoric acid medium was undertaken. Spectrographic analysis of the column effluent was attractive because the rapid simultaneous determination of many elements could be made and radioacthe tracers \\-ere not necessary. Results presented in this surrey were obtained by visually estimating quantities of each element found in effluent fractions from their spectral intensity. The individual determinations, while not estremely accurate in themselves, were used t o draw elution curves representing the flon- from the column; because the beginning and the end of the curves could be readily seen, the peak could usually be estimated to m-ithin several
column volumes, and approximations made as to the general shape of the curve. The principal advantage of spectrographic analysis is demonstrated in the fact that over 60 element species were studied in a few months. MATERIALS AND PROCEDURE
Resin. -4 large batch of Dowex 1-X10, 200-mesh anion exchange resin n-as converted from the chloride form b,y washing with hydrofluoric acid until a silver nitrate test for chloride 11as negative. Analytical grade resin (supplied by Bio-Rad Laboratories, Inc., Berkeley, Calif.) was found to be sufficiently free of foreign material to require no further purification. The rated capacity was 3 meq. per gram and the approximate density, determined from the displacement of benzene by the oren-dried resin, was 1.67 grams per cc. Columns. Eight polyethylene columns inch in inside diameter, each containing 2.8 grams of oven-dried resin, were used for the initial survey. The resin was introduced to t h e columns from a dilute hydrofluoric acid slurry and retained by finely ground polyethylene packed in the lower tip. Each column \\as used for a selected molarity of hydrofluoric acid throughout. Concentrations of 24, 20, 16, 12,
